Disulfide bond formation in a protein is an important post-translational modification affecting its tertiary structure. It is important to know, how many of the cysteines are free and how many are tied up as disulfide bonds. The conventional analysis involves chemical marking or labeling of the cysteines that are free followed by reduction and similar chemical marking of the nascent sulfhydryls released from reduction of disulfide bonds. The reduced denatured protein is then digested enzymatically followed by separation of the resulting fragments, the huge proportion of which contain no information regarding the cysteines but which nevertheless must be separated and identified in the search for cystinyl peptide fragments to be identified by mass spectrometry. In an effort to simplify this methodology, we have been investigating the use of nitrothiocyanobenzoic acid (NTCB) because it chemically cleaves the peptide chain on the N-terminal side of a free cysteine. Such chemical degradation provides a simplified mixture of fragments compared to those released trypsin. Our simplified assay is amenable to analysis by MALDI at the picomole level. We have demonstrated the utility of this approach with several model proteins ranging in molecular weight up to 15 kDa and containing as many as seven cysteines in a variety of situations in which some are tied up as cystines.